The preparation of a new type of inhibitor of some of the key folate enzymes is based on structures proposed for the intermediates involved in the reactions that these enzymes catalyze. The new compounds, 5-, 10-, and bridged 5, 10-substituted tetrahydrofolic acids, will be designed to act either as analogs of the covalent linked substrate-cofactor complex or as analogs of the cofactor. In the former, the affinity of the analog for the enzyme is expected to be greater than the individual affinities of the natural cofactor and substrate. In the latter, inhibition will result from the formation of a covalent linkage between the pseudo cofactor and substrate when both are bound to the enzyme. These tetrahydrofolic acid derivatives will be evaluated in cell-free systems for enzyme inhibition and in intact cells by procedures designed to detect blockages of the folate enzymes. In addition, cytotoxicity in the KB cell culture screen and activity against lymphocytic leukemia P388 in mice will be determined. Furthermore, the compounds showing activity in the above systems will be tested for activity in tumors resistant to the conventionally used agents such as methotrexate. The discovery of inhibitors of the folate enzymes other than dihydrofolate reductase might result in the development of more effective methods for the treatment of cancer.